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RESTRICTION MAPPING


RESTRICTION MAPPING


Introduction


Restriction mapping is a molecular biology technique used to determine the relative positions of restriction enzyme recognition sites on a DNA molecule. It involves digestion of DNA with one or more restriction endonucleases followed by analysis of fragment sizes using agarose gel electrophoresis.
Restriction mapping is essential for DNA characterization, cloning strategies, gene localization, and genome analysis.

Definition


Restriction mapping is the process of identifying the number, order, and distances between restriction enzyme cleavage sites within a DNA fragment by analyzing the pattern of fragments generated after enzymatic digestion.


Principle


Restriction enzymes cut DNA at specific palindromic nucleotide sequences. When DNA is digested with:
Single restriction enzyme → produces fragments based on its recognition sites
Multiple restriction enzymes → produces fragments whose sizes reveal the relative positions of sites
By comparing fragment sizes from single and double digestions, a physical map of restriction sites can be constructed.


Types of Restriction Mapping


Single-enzyme restriction mapping
Double-enzyme restriction mapping
Partial digestion mapping
Restriction mapping using cloned DNA
Requirements
DNA sample (linear or circular)
Restriction endonucleases
Appropriate buffers
Agarose gel electrophoresis apparatus
DNA ladder (molecular weight marker)
Staining dye (Ethidium bromide / SYBR Safe)


Methodology


1. Isolation of DNA
DNA is extracted from cells using standard isolation techniques.
Purified DNA is quantified and checked for integrity.


2. Restriction Enzyme Digestion


DNA is divided into separate tubes:
Tube 1: Digestion with Enzyme A
Tube 2: Digestion with Enzyme B
Tube 3: Double digestion (A + B)
Reaction conditions:
Optimal temperature (usually 37°C)
Correct buffer and incubation time


3. Agarose Gel Electrophoresis


Digested DNA fragments are separated based on size
Smaller fragments move faster than larger ones
DNA ladder used to estimate fragment sizes

4. Analysis of Fragment Sizes


Measure the size of fragments from gel
Compare single and double digestion patterns
Identify overlapping fragments

5. Construction of Restriction Map


Arrange fragments in correct order
Mark enzyme cutting sites
Distance between sites calculated from fragment sizes
Final diagram represents the restriction map


Example of Restriction Mapping
A linear DNA of 10 kb:
Enzyme A cuts into 6 kb + 4 kb
Enzyme B cuts into 7 kb + 3 kb
A + B digestion gives 4 kb, 3 kb, 2 kb, 1 kb
Using overlap analysis, restriction sites are mapped on DNA.


Partial Digestion Mapping


DNA is exposed to limited enzyme concentration or time
Produces overlapping fragments
Useful for determining order of closely spaced sites
Important for large DNA molecules
Restriction Mapping of Circular DNA
Plasmids produce different fragment patterns
Single cut → linear DNA
Multiple cuts → multiple fragments
Map is circular rather than linear
Applications of Restriction Mapping
Gene cloning and vector construction
Identification of recombinant DNA
Verification of plasmid inserts
Genome organization studies
Comparative DNA analysis
Mutation detection
Forensic and diagnostic studies


Advantages


Simple and cost-effective
High specificity
Useful for DNA characterization
Essential for cloning experiments

Limitations


Limited resolution for very large DNA
Requires known restriction enzymes
Time-consuming for complex genomes
Cannot detect single nucleotide changes
Modern Alternatives
DNA sequencing
Optical mapping
Whole genome restriction mapping (WGRM)


Conclusion


Restriction mapping is a fundamental molecular biology technique that provides detailed information about the structure and organization of DNA molecules. Though largely replaced by sequencing in advanced research, it remains indispensable in cloning, plasmid analysis, and teaching laboratories.


Restriction mapping is used to determine:
A) DNA sequence
B) Position of restriction sites on DNA ✅
C) Protein structure
D) RNA expression

Restriction enzymes are also called:
A) DNA ligases
B) Endonucleases ✅
C) Polymerases
D) Helicases

Most restriction enzymes recognize:
A) Random sequences
B) Palindromic sequences ✅
C) Only AT-rich sequences
D) Only GC-rich sequences


A palindromic sequence reads the same:
A) 5′ → 3′ on one strand only
B) 5′ → 3′ on both strands ✅
C) 3′ → 5′ on both strands
D) Randomly


Which type of DNA can be mapped using restriction mapping?
A) Only linear DNA
B) Only circular DNA
C) Both linear and circular DNA ✅
D) Only mitochondrial DNA
Enzyme Digestion
Single digestion of DNA with one enzyme gives:
A) No fragments
B) Fragment sizes based on enzyme sites ✅
C) A complete sequence
D) Only one fragment regardless of cuts
Double digestion involves:
A) Two enzymes in one tube or sequentially ✅
B) Two DNA samples with same enzyme
C) Two buffers
D) DNA denaturation
Partial digestion is used to:
A) Cut DNA completely
B) Determine order of closely spaced sites ✅
C) Produce only linear DNA
D) Remove methylation

Which factor affects restriction enzyme activity?
A) Temperature 
B) Buffer
C) Incubation time
D) All of the above ✅
EcoRI is an example of:
A) Type I restriction enzyme
B) Type II restriction enzyme ✅
C) Type III restriction enzyme
D) DNA polymerase
Electrophoresis & Fragment Analysis
DNA fragments are separated by:
A) SDS-PAGE
B) Agarose gel electrophoresis ✅
C) Centrifugation
D) Chromatography
Smaller DNA fragments move:
A) Slower
B) Faster ✅
C) At same speed as large
D) Randomly
A DNA ladder is used to:
A) Cut DNA
B) Stain DNA
C) Estimate fragment sizes ✅
D) Denature DNA
Agarose gel concentration is chosen based on:
A) Buffer type
B) DNA size ✅
C) Restriction enzyme type
D) Incubation temperature
Ethidium bromide binds to:
A) Proteins
B) RNA
C) DNA ✅
D) Lipids
Mapping Techniques
Restriction mapping produces:
A) DNA sequence
B) Fragment length polymorphism
C) Physical map of restriction sites ✅
D) Gene expression profile
Circular DNA single cut produces:
A) Linear DNA ✅
B) Multiple fragments
C) No change
D) RNA
Partial digestion produces:
A) Overlapping fragments ✅
B) Only single fragment
C) Only circular DNA
D) DNA methylation
Double digestion fragments help in:
A) Determining fragment color
B) Determining order of restriction sites ✅
C) Determining sequence
D) Producing mRNA
Overlapping fragment analysis is used in:
A) Single digestion mapping
B) Double digestion mapping ✅
C) RNA analysis
D) Protein purification
Applications
Restriction mapping is useful in:
A) Cloning ✅
B) Protein synthesis
C) RNA splicing
D) Lipid analysis
It helps verify:
A) PCR amplification
B) Plasmid insert correctness ✅
C) mRNA expression
D) DNA replication
Restriction mapping can detect:
A) Single nucleotide polymorphisms (SNPs) ✅
B) Protein folding errors
C) RNA secondary structure
D) Lipid modifications
It is used in forensic studies for:
A) Protein expression
B) DNA fingerprinting ✅
C) RNA profiling
D) Cloning vectors only
Physical maps are useful for:
A) Sequencing strategy design ✅
B) Protein crystallization
C) RNA translation
D) Lipid analysis
Advanced Concepts
Type II restriction enzymes:
A) Cut at specific recognition site ✅
B) Cut randomly
C) Require ATP
D) Only work on RNA
Restriction map distance is measured in:
A) bp (base pairs) ✅
B) nm
C) μm
D) kDa
Which of these produces sticky ends?
A) EcoRI ✅
B) SmaI
C) AluI
D) HindIII
Blunt-end cutters:
A) Produce overhangs
B) Produce blunt ends ✅
C) Only digest RNA
D) Only digest protein
Overhangs are useful for:
A) Ligase-independent cloning
B) Facilitating ligation ✅
C) DNA methylation
D) Gel staining
Experimental Considerations
Incomplete digestion occurs due to:
A) Low enzyme activity ✅
B) Excess buffer
C) High temperature
D) Overstaining
DNA concentration affects:
A) Fragment mobility
B) Digestion efficiency ✅
C) DNA ladder color
D) DNA methylation
Restriction mapping cannot detect:
A) Enzyme cutting sites
B) Large deletions
C) Single base mutations without a site change ✅
D) Plasmid insertions
Restriction mapping is less effective for:
A) Small plasmids
B) Large genomic DNA ✅
C) Linear DNA
D) Viral DNA
Gel electrophoresis separates fragments based on:
A) Charge only
B) Size and charge ✅
C) Sequence
D) Enzyme used
Problem-Solving MCQs
A 10 kb DNA digested by enzyme A gives 6 kb + 4 kb; enzyme B gives 7 kb + 3 kb; double digestion gives 4 kb, 3 kb, 2 kb, 1 kb. What does this show?
A) Sites overlap ✅
B) Single site
C) No cuts
D) Circular DNA
Partial digestion is used when:
A) Sites are far apart
B) Sites are very close ✅
C) DNA is RNA
D) For protein mapping
In double digestion, fragment patterns help:
A) Sequence DNA
B) Map site order ✅
C) Cut RNA
D) Denature proteins
Restriction mapping can identify:
A) Protein folding errors
B) DNA mutations that alter sites ✅
C) RNA splicing
D) Lipid composition
EcoRI cuts:
A) GAATTC ✅
B) GGCC
C) CCCGGG
D) ATGC
Practical & Conceptual
Which is true about plasmid restriction mapping?
A) Single cut linearizes plasmid ✅
B) Plasmid cannot be mapped
C) Only double cuts work
D) Ends remain circular
Agarose percentage affects:
A) DNA digestion
B) Fragment resolution ✅
C) Enzyme recognition
D) DNA methylation
Molecular weight marker helps:
A) Identify enzyme
B) Estimate fragment size ✅
C) Change DNA sequence
D) Linearize plasmid
Restriction maps are also called:
A) Physical maps ✅
B) Genetic maps
C) RNA maps
D) Protein maps
Type I enzymes:
A) Cut at recognition site
B) Cut randomly far from site ✅
C) Require no cofactors
D) Only work on RNA
Applications & Limitations
Limitation of restriction mapping:
A) Cannot detect very large DNA fragments easily ✅
B) Cannot identify enzyme
C) Cannot cut RNA
D) Cannot visualize fragments
Advantage of restriction mapping:
A) Fast DNA sequencing
B) Characterizes DNA structure ✅
C) Protein analysis
D) RNA profiling
Restriction mapping is important in cloning to:
A) Amplify RNA
B) Verify recombinant DNA ✅
C) Identify proteins
D) Study lipids
Physical maps can be converted to:
A) RNA sequence
B) DNA sequence ✅
C) Protein sequence
D) Lipid profile
Modern alternative to restriction mapping:
A) Optical mapping ✅
B) SDS-PAGE
C) Northern blotting
D) Western blotting

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